Protein Aggregation Domains and Methods of Use Thereof

Abstract

Using the Sup35 prion proteins of two distantly related yeast species, it is established that prion replication is initiated by small elements of primary sequence, which can be identified using arrays of short peptides. Subtle differences in replication elements govern the formation of distinct aggregate conformations (prion strains) and also determine their species-specific seeding activities. A Sup35 chimera that promiscuously forms prions in more than one species does so by virtue of carrying the replication element of each species. Mutations or conditions that cause the chimera to assemble into distinct prion strains favor recognition of distinct replication elements. Therefore, subtle differences in small sequences that constitute prion replication elements encode important determinants of prion propagation and transmission. The protein aggregation domains, methods for identification thereof, and polypeptides and higher order aggregates including the protein interaction domains, as well as arrays including peptides derived from an aggregation-prone polypeptide are provided.

1 . A peptide comprising at least 15 contiguous amino acids located between amino acids 1-40 of Sc Sup35, wherein said contiguous amino acids include amino acids 18-22 of Sc Sup35 and assemble with full length Sc Sup35 to form a higher ordered aggregate, and wherein the sequence of said polypeptide does not contain more than 50 contiguous amino acids of the sequence of Sc Sup35 outside the region between amino acids 1-40 of Sc Sup35. 
     
     
         2 . The peptide of  claim 1 , wherein the peptide does not contain more than 20 contiguous amino acids of the sequence of Sc Sup35 outside the region between amino acids 1-40 of Sc Sup35. 
     
     
         3 . The peptide of  claim 1 , wherein the peptide is located between amino acids 8-40 of Sc Sup35. 
     
     
         4 . The peptide of  claim 1 , wherein the peptide is located between amino acids 8-32 of Sc Sup35. 
     
     
         5 . The polypeptide of  claim 1 , wherein the amino acid sequence comprises or consists of amino acids 10-29 of Sc Sup35. 
     
     
         6 . The polypeptide of  claim 1 , wherein the amino acid sequence comprises or consists of amino acids 11-30 of Sc Sup35. 
     
     
         7 . The peptide of  claim 1 , wherein the polypeptide is between 10 and 50 amino acids in length. 
     
     
         8 . The peptide of  claim 1 , wherein the peptide is between 15 and 30 amino acids in length. 
     
     
         9 . A peptide at least 90% identical to the peptide of  claim 1 . 
     
     
         10 . A peptide whose sequence differs by not more than 2 amino acid insertions, deletions, or substitutions from that of the peptide of  claim 1 . 
     
     
         11 . A polypeptide whose amino acid sequence comprises a first portion that comprises the peptide of any one of  claims 1 - 10 . 
     
     
         12 . The peptide of  claim 1 , further comprising a second portion, wherein the second portion has a biological or chemical activity of interest or comprises a detectable, selectable, or reactive moiety. 
     
     
         13 . A higher order aggregate comprising a peptide of  claim 1 . 
     
     
         14 . The higher order aggregate of  claim 13 , wherein said higher order aggregate is a fibril. 
     
     
         15 . A solid support having the peptide of  claim 1  covalently or noncovalently attached thereto. 
     
     
         16 . A peptide comprising at least 15 contiguous amino acids located between amino acids 59-86 of Ca Sup35, wherein the contiguous amino acids include amino acids 69-76 of Ca Sup35 and assemble with full length Ca Sup35 to form a higher ordered aggregate, and wherein the sequence of said polypeptide does not contain more than 50 contiguous amino acids of the sequence of Ca Sup35 outside the region between amino acids 59-86 of Sc Sup35. 
     
     
         17 . The peptide of  claim 1 , wherein the peptide does not contain more than 20 contiguous amino acids of the sequence of Ca Sup35 outside the region between amino acids 59-86 of Sc Sup35. 
     
     
         18 . The peptide of  claim 16  wherein the polypeptide is between 10 and 50 amino acids in length. 
     
     
         19 . The peptide of  claim 16 , wherein the peptide is between 15 and 30 amino acids in length. 
     
     
         20 . A peptide at least 90% identical to the peptide of  claim 16 . 
     
     
         21 . A peptide whose sequence differs by not more than 2 amino acid insertions, deletions, or substitutions from that of the peptide of  claim 16 . 
     
     
         22 . A polypeptide whose amino acid sequence comprises a first portion that comprises the peptide of any one of  claims 16 - 21 . 
     
     
         23 . The polypeptide of  claim 16 , further comprising a second portion, wherein the second portion has a biological or chemical activity of interest or comprises a detectable, selectable, or reactive moiety. 
     
     
         24 . A higher order aggregate comprising a peptide of  claim 16 . 
     
     
         25 . The higher order aggregate of  claim 24 , wherein said higher order aggregate is a fibril. 
     
     
         26 . A solid support having the peptide of  claim 16  covalently or noncovalently attached thereto. 
     
     
         27 . A collection comprising at least 10 different peptides, wherein the peptides are fragments of a polypeptide, wherein the polypeptide is a polypeptide that misfolds or spontaneously aggregates into a higher order structure under appropriate conditions. 
     
     
         28 . The collection of  claim 27 , wherein the peptides scan across between 20% and 100% of the polypeptide and wherein the N-terminal amino acids of the peptides are located between 1 and 10 amino acids from each other within the polypeptide sequence. 
     
     
         29 . The collection of  claim 27 , wherein the peptides are derived from a polypeptide selected from the group consisting of: Sup35 proteins, Ure2 proteins, New1 proteins, Rnq1 proteins, mammalian prion proteins, amyloid precursor protein, Aβ40, Aβ42, immunoglobulin (Ig) light chain, serum amyloid A, wild type or variant transthyretin, lysozyme, BnL, cystatin C, β2-microglobulin, apoliprotein A1, gelsolin mutants, lactotransferrin, islet amyloid polypeptide, fibrinogen, prolactin, insulin, calcitonin, atrial natriuretic factor, α-synuclein, Huntingtin, superoxide dismutase, and α1-chymotrypsin. 
     
     
         30 . An array comprising a plurality of peptides, wherein the peptides are fragments of a polypeptide, wherein the polypeptide is a polypeptide that misfolds or spontaneously aggregates into a higher order structure under appropriate conditions. 
     
     
         31 . The array of  claim 30 , wherein the peptides are derived from a polypeptide selected from the group consisting of: Sup35 proteins, Ure2 proteins, New1 proteins, Rnq1 proteins, mammalian prion proteins, amyloid precursor protein, Aβ40, Aβ42, immunoglobulin (Ig) light chain, serum amyloid A, wild type or variant transthyretin, lysozyme, BnL, cystatin C, β2-microglobulin, apoliprotein A1, gelsolin mutants, lactotransferrin, islet amyloid polypeptide, fibrinogen, prolactin, insulin, calcitonin, atrial natriuretic factor, α-synuclein, Huntingtin, superoxide dismutase, and α1-chymotrypsin. 
     
     
         32 . A method of forming a higher ordered aggregate comprising the steps of:
 providing a composition comprising (a) a peptide comprising a protein aggregation domain and (b) a polypeptide comprising the protein aggregation domain; and   maintaining the composition for a time sufficient for formation of a higher ordered aggregate.   
     
     
         33 . The method of  claim 32 , wherein the peptide is between 15 and 50 amino acids in length. 
     
     
         34 . The method of  claim 32 , wherein the polypeptide is selected from the group consisting of: Sup35 proteins, Ure2 proteins, New1 proteins, Rnq1 proteins, mammalian prion proteins, amyloid precursor protein, Aβ40, Aβ42, immunoglobulin (Ig) light chain, serum amyloid A, wild type or variant transthyretin, lysozyme, BnL, cystatin C, β2-microglobulin, apoliprotein A1, gelsolin mutants, lactotransferrin, islet amyloid polypeptide, fibrinogen, prolactin, insulin, calcitonin, atrial natriuretic factor, α-synuclein, Huntingtin, superoxide dismutase, and α1-chymotrypsin. 
     
     
         35 . The method of  claim 32 , wherein the peptide is attached to a solid support. 
     
     
         36 . The method of  claim 35 , further comprising removing the higher ordered aggregate from the solid support. 
     
     
         37 . A method of identifying an aggregation domain of a polypeptide comprising the steps of:
 providing an array comprising a plurality of peptides, wherein the peptides are fragments of a polypeptide that spontaneously aggregates into a higher order structure under appropriate conditions;   contacting the array with the polypeptide; and   identifying a peptide to which the polypeptide binds, thereby identifying an aggregation domain of the polypeptide.   
     
     
         38 . The method of  claim 37 , wherein the polypeptide is selected from the group consisting of Sup35 proteins, Ure2 proteins, New1 proteins, Rnq1 proteins, mammalian prion proteins, amyloid precursor protein, Aβ40, Aβ42, immunoglobulin (Ig) light chain, serum amyloid A, wild type or variant transthyretin, lysozyme, BnL, cystatin C, β2-microglobulin, apoliprotein A1, gelsolin mutants, lactotransferrin, islet amyloid polypeptide, fibrinogen, prolactin, insulin, calcitonin, atrial natriuretic factor, α-synuclein, Huntingtin, superoxide dismutase, and α1-chymotrypsin. 
     
     
         39 . The method of  claim 37 , wherein the polypeptide is one whose misfolding or aggregation is implicated in mammalian disease. 
     
     
         40 . A method of identifying a candidate agent for modulating protein aggregation comprising:
 (i) providing a composition comprising an aggregation-prone polypeptide, a test agent, and a peptide derived from the aggregation-prone polypeptide, wherein the peptide is capable of binding to the aggregation-prone polypeptide in the absence of the test agent; and   (ii) identifying the agent as a candidate agent for modulating protein aggregation if presence of the test agent alters the extent or rate of binding of the peptide and the polypeptide.   
     
     
         41 . A method for identifying a candidate agent for inhibiting protein aggregation comprising:
 (i) providing a composition comprising an aggregation-prone polypeptide, a test agent, and a peptide derived from the aggregation-prone polypeptide, wherein the peptide is capable of binding to the aggregation-prone polypeptide in the absence of the test agent; and   (ii) identifying the agent as a candidate agent for inhibiting protein aggregation if presence of the test agent reduces the binding of the peptide and the polypeptide.